Enamel fluorosis is a defect in enamel development that occurs after exposure to excess fluoride. Fluorotic enamel is more porous, and contains more proteins than sound enamel. The mechanisms by which fluoride alters enamel formation remain incompletely understood. In this competing renewal, we propose to build on work that we have completed in previous grant cycles, to test a new paradigm that defines fluorosis as resulting from matrix related changes that occur as fluoride is incorporated into the forming enamel mineral. Specifically, we propose that incorporation of fluoride into the developing enamel crystals alters matrix/protein/proteinase interactions, and also can reduce matrix pH secondary to more rapid mineral formation. These pH-related effects alter ameloblast function and modulation, and enamel maturation. Furthermore, we propose that when matrix proteins or mineralizing apatite are not available to bind fluoride ions (ie presecretory stage), fluoride can have a direct effect on gene expression. A thorough understanding of the mechanisms by which fluoride can alter enamel formation will allow us to develop strategies to minimize the negative effects of fluoride while enhancing the use of fluoride for caries prevention We will investigate these potential mechanisms of dental fluorosis through the following three specific aims. Specific Aim 1: To determine the role of fluoride incorporation into the growing enamel mineral on apatite/ protein interactions and amelogenin hydrolysis. Mass spectrometry will be used to determine how fluoride-containing apatites alter amelogenin hydrolysis both in vitro and in vivo. Specific Aim 2: To determine how fluoride-related changes in enamel matrix pH affects ameloblast modulation and mineral deposition. Mouse models with a reduced capacity to buffer enamel matrix related pH changes will be used to correlate changes in matrix pH to ameloblast modulation and enamel mineralization. Specific Aim 3: To determine the effects of fluoride on the expression of genes related to enamel matrix protein deposition and pH regulation. Laser capture micro-dissection will be used to separately collect presecretory, secretory and maturation stage ameloblasts from mice with and without fluoride exposure for either whole transcript comparisons, or qPCR analysis of genes that modulate pH in the developing enamel matrix. These studies will be done with an exceptional team of international investigators, and we anticipate that at the completion of this grant cycle, that we will have a thorough understanding of how fluoride exposure during enamel development results in fluorosis.